The structures, organization and functions of cytoskeletal filaments, membranes, extracellular matrices and other biological systems depend on specific interactions between many macromolecules. A major problem which faces biologists is to identify these specific interactions. One approach is to isolate macromolecules and examine their interactions in vitro, but this approach is limited by the difficulty of extrapolating results to the situation in living cells. The in vitro approach needs to be supplemented with methods for analyzing protein-protein interactions in situ.
Attention is directed to an article by K. Peters and F. M. Richards entitled, "Chemical Cross-Linking: Reagents and Problems in Studies of Membrane Structure" in 46 Annual Review of Biochemistry 523-551 (1977) wherein the authors describe the state-of-the-art in studying protein-protein interactions in situ using chemical cross-linking reagents. Attention is also directed to an article by T. H. Ji entitled "A Novel Approach to the Identification of Surface Receptors" in 252 Journal of Biological Chemistry 1566-1570 (1977) and an article by D. J. Kiehm and T. H. Ji entitled "Photochemical Cross-Linking of Cell Membranes" in 252 Journal of Biological Chemistry 8524-8531 (1977), wherein a cleavable photosensitive cross-linking reagent is disclosed.
In general bifunctional cross-linking reagents serve to join a first material, i.e. a protein, with a second material, for example, an interacting protein. The cross-linked complexes are analyzed by gel electrophresis and this data leads to hypotheses on the interactions or proximity of such materials in biological systems. A major problem with this technique is the limitation of gel electrophoresis; large complexes formed by macromolecules can not be effectively taken up into the gels and electrophoretically distinguished.
Cleavable bifunctional crosslinkers, as described in the Peters article cited above, are also known. A cleavable crosslinker permits two-way electrophoresis. The complexes are first subjected to electric field induced migration in one direction and then reduced to cleave the crosslinker and subjected to migration in a second, typically perpendicular, direction. This cleaving technique is also limited since the complexity of the patterns often precludes meaningful conclusions.
Heterobifunctional crosslinkers are designed for two-stage crosslinking where a material of interest, for example a hormone, can be isolated, linked to one end of the reagent and then introduced in situ for reaction at the other end. This technique typically involves the use of photosensitive crosslinkers; a protein, with one end of the crosslinker attached thereto, is introduced into the system and the other end of the crosslinker is unreactive until it is photolyzed. Analysis by electrophoresis is simplified because one component of the complex is known: the introduced protein.
Related to crosslinking reagents are affinity probes. In such probes, a radioactively-labelled molecule or protein is attached to a reagent and introduced into the biological system. Complexes formed by the probe can be identified by their radioactivity and analyzed by electrophoresis.
There exists a need for better cross-linking reagents, particularly cleavable heterobifunctional cross-linkers. Moreover, it would be advantageous to have a crosslinking reagent that can donate an identifiable label which will remain on the second material or protein after cleavage. This "donation" property would permit the complexes formed by the crosslinker to be reduced to their constituent parts prior to electrophoresis thus allowing simplified analysis of those constituents which are labelled by the donating crosslinker.